The objective of this proposal is to provide a systematic study of relations between membrane stability, membrane transport and the binding of Ca and mg to specific membrane sites. Membrane bound Ca and mg will be altered by subjecting both red cells and red ghosts to various physicochemical treatments (e.g. enzymes, chelating agents, low ionic strength). Water non-electrolyte, and salt transport will be measured and the relevant parameters will be related to measured amounts of Ca (and/or mg that remain bound to the membrane). Further studies will be undertaken to identify critical binding sites for divalent cations which are involved in specific transport processes. Differences between location on inner and outer membrane surfaces as well as chemical identity of these sites will be sought. The use of fluorescent probes as an indicator of changes in membrane structure (e.g. packing density of phospholipid aggregates, changes in protein conformation) will be explored in an effort to correlate these changes with divalent cation binding and transport. Relating patterns of permeability changes (using a variety of different solute probes) to binding and fluorescence studies will provide a better understanding of the relation of membrane structure to membrane transport.